Prenylated polyphenols have attracted a lot of interest because of their significant pharmacological activities. The chemoenzymatic synthesis of prenylated polyphenols using prenyltransferase (PT) has been regarded as a promising alternative to chemical synthesis or plant-based production. Psoralea corylifolia L. with abundance of diversified prenylated polyphenol compounds is an ideal material for the discovery of prenyltransferases available in synthetic biology. In this study, we investigated the transcriptomic data from P. corylifolia for prenyltransferases (PcPTs) involved in the biosynthesis of prenylated phenols. Among 11 PcPTs, our yeast expression in vitro survey showed that PcPT11 was able to utilize 23 substrates, including 22 flavonoids and 1 coumarin, and was the one able to use the most substrates. PcPT11 acts in a highly regio-specific way to prenylate flavonoids at the C-6 position to produce compounds with important pharmacological effects such as corylifolinin, wighteone, and bavachin, which are the major ingredients in P. corylifolia. Finally, PcPT11 exhibited strong expression in fruits and leaves, and a GFP-PcPT11 fusion protein was shown to be localized in chloroplasts when expressed in Nicotiana benthamiana. Our study laid a foundation for understanding the biosynthetic pathway of prenylated flavonoids in P. corylifolia, and demonstrated that PcPT11 is a valuable enzyme to produce these pharmaceutically important prenylated polyphenols through biosynthetic approaches.

植物类黄酮化合物是一类重要的天然产物,通常以糖苷的形式存在.尿苷二磷酸糖基转移酶(uridine diphosphate glycosyltransferase,UGT)能够对类黄酮进行糖基化修饰,形成种类丰富的类黄酮糖苷,是许多药用植物中的类黄酮药用活性成分.近年来,随着越来越多的植物基因组被解析,大量参与类黄酮合成的糖基转移酶得以鉴定.本文首先简述了植物UGT的结构特征和家族分类,然后详细综述了植物类黄酮UGT的研究进展,对处于不同家族中的植物类黄酮UGT的修饰位点特异性、以及糖供体和糖受体的特异性进行了全面的归纳和总结,以期为植物类黄酮UGT的结构与功能相关性研究及新植物类黄酮UGT的发掘与鉴定研究奠定基础.

Epimedium pubescens Maxim. is a well-known traditional Chinese medicinal herb with flavonol glycosides as the major pharmaceutically active compounds. UDP-glycosyltransferases (UGTs) are a group of enzymes responsible for the glycosylation of flavonoid glycosides. In this study, a genome-wide analysis was performed to identify UGT family genes in E. pubescens. As a result, a total of 339 putative UGT genes were identified, which represents the largest UGT gene family known thus far, implying a significant expansion of the UGT gene family in E. pubescens. All EpUGTs were unevenly distributed across six chromosomes, and they were classified into 17 major groups. The expression profiles showed that UGT genes were differentially expressed in roots, leaves, flowers, shoots and fruits. In particular, several EpUGTs were highly induced by high light intensity, which was consistent with the accumulation level of bioactive flavonoids in E. pubescens. Six UGT79 genes that were preferentially expressed in roots or leaves were successfully expressed in E. coli, and only the recombinant EpGT60 protein was found to be active toward 8-prenylkaempferol and icaritin to produce the key bioactive compounds baohuoside II and baohuoside I. The optimal temperature, pH, km and Vmax were determined for the recombinant EpGT60 protein. In addition, expression of recombinant EpGT60 in E. coli cell culture led to successful production of baohuoside II when fed 8-prenylkaempferol. Our study provides a foundation for further functional characterization of UGT genes in E. pubescens and provides key candidate genes for bioengineering bioactive flavonoids in E. pubescens.

Prenylated flavonol glycosides in Epimedium plants, as key medicinal components, are known to have great pharmaceutical activities for human health. Among the main prenylated flavonol glycosides, the modification mechanism of different sugar moieties is still not well understood. In the current study, a novel prenylated flavonol rhamnoside xylosyltransferase gene (EpF3R2″XylT) was cloned from E. pubescens, and the enzymatic activity of its decoding proteins was examined in vitro with different prenylated flavonol rhamnoside substrates and different 3-O-monosaccharide moieties. Furthermore, the functional and structural domains of EpF3R2″XylT were analyzed by bioinformatic approaches and 3-D protein structure remodeling. In summary, EpF3R2″XylT was shown to cluster with GGT (glycosyltransferase that glycosylates sugar moieties of glycosides) through phylogenetic analysis. In enzymatic analysis, EpF3R2″XylT was proven to transfer xylose moiety from UDP-xylose to prenylated flavonol rhamnoside at the 2″-OH position of rhamnose. The analysis of enzymatic kinetics showed that EpF3R2″XylT had the highest substrate affinity toward icariin with the lowest Km value of 75.96 ± 11.91 mM. Transient expression of EpF3R2″XylT in tobacco leaf showed functional production of EpF3R2″XylT proteins in planta. EpF3R2″XylT was preferably expressed in the leaves of E. pubescens, which is consistent with the accumulation levels of major prenylflavonol 3-O-triglycoside. The discovery of EpF3R2″XylT will provide an economical and efficient alternative way to produce prenylated flavonol trisaccharides through the biosynthetic approach.